1. Field of the Invention.
The invention relates to an assay and a device for detecting and measuring the activities and concentrations of at least two proteins having similar properties or overlapping properties. In particular, the invention relates to an assay and a device for detecting and measuring the activities and concentrations of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), or both in a sample.
2. Description of the Related Art.
Cholinesterases (ChEs) are highly polymorphic carboxylesterases of broad substrate specificity, involved in the termination of neurotransmission in cholinergic synapses and neuromuscular junctions. Some ChEs terminate the electrophysiological response to the neurotransmitter acetylcholine by rapidly degrading it, while the precise function of others is unknown. ChEs are classified into acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) according to their substrate specificity and sensitivity to selective inhibitors. See Massoulie, J., et al., (1982) Ann. Rev. Neurosci. 5:57-106, which is incorporated herein by reference.
AChE is one of nature""s most elegantly engineered proteins. AChE accelerates the hydrolysis of acetylcholine, a neurotransmitter, at nervexe2x80x94nerve and neuromuscular junctions. BChE is found in mammalian blood, plasma, liver, pancreas, intestinal mucosa and the white matter of the central nervous system. BChE is also known as pseudocholinesterase and is sometimes referred to as serum cholinesterase as opposed to red blood cell cholinesterase, true cholinesterase, or AChE. BChE catalyzes the hydrolysis of a number of choline esters.
BChE also degrades cocaine ingested by a subject. Generally, cocaine is well tolerated by the majority of the population. However, acute cocaine abuse is related to a small incidence of sudden death. See Clouet, D. et al., Mechanisms of Cocaine Abuse and Toxicity, NIDA Research Monograph 88; and Johanson, C. and Fischman, M. W., (1989) Pharmacol. Rev. 41:3, which are both incorporated herein by reference. Although the physiological basis for sudden death due to acute cocaine abuse is not known, it is possible that abnormal BChE activity and amounts may contribute to a subject""s sensitivity to cocaine. See Stewart, D. J. et al., (1979) Clin. Pharmacol. Ther. 25:464; Jatlow, P., (1979) Anesth. Anag., 58:235; Anton, A. H., (1988) Drug Intell. Clin. Pharm. 22:914; and Devenyl, P., (1989) Ann. Int. Med. 110:167, all of which are incorporated herein by reference.
BChE hydrolyzes and inactivates muscle relaxants such as succinylcholine and related anesthetics. About 5% of the population have an abnormal genotype for BChE, which results in a severe deficiency in BChE activity and amounts. When a subject having an abnormal genotype for BChE is administered succinylcholine for inducing general anesthesia prior to surgery, the subject may experience a prolonged apnea as compared to a subject having a normal genotype for BChE during which the subject is unable to breathe and must be artificially ventilated until the succinylcholine is degraded by secondary mechanisms. As this condition is a potentially life-threatening situation, a subject may be screened for abnormal BChE activity and amounts and then administered BChE before, during, or after general anesthesia. Clearly, it would be desirable to periodically measure the subject""s amounts, activities, and sensitivities of BChE, AChE, or both.
Succinylcholine sensitivity may also result from an abnormal BChE concentration or activity caused by pregnancy, diseases such as liver disease and hepatitis, or medications. See Wildsmith, J. A. W., (1972) Anesthesia 27:90; Weissman, D. B., et al., (1983) J., Anesth. Analg. 62:444; Singh, D.C., et al., (1976) J. Ind. Med. Assoc. 66:49; and Foldes, F. F., Enzymes in Anesthesiology, (1978) Springer-Verlag, NY, all of which are herein incorporated by reference.
As succinylcholine and cocaine sensitivity and other diseases such as Alzheimer""s disease, glaucoma, and myasthenia gravis or any other such disease may be treated by regulating the concentrations or activities of AChE, BChE, or both, it would be desirable to detect, measure and monitor the concentrations and activities of AChE and BChE.
Nerve agents, chemical warfare agents, organophosphates (OPs), pesticides, insecticides, and other such noxious chemicals exert their toxic effects by inhibiting AChE, BChE, or both. Plasma BChE and erythrocyte AChE provide some protection to synaptic AChE from these neurotoxins by scavenging free circulating AChE toxins, BChE toxins, or both prior to absorption into the central and peripheral nervous systems. Only the non-scavenged neurotoxins are capable of attacking synaptic AChE. Therefore, a subject""s susceptibility to these neurotoxins may be determined by measuring the concentrations and activities of AChE and BChE in the subject. Additionally, exposure to these neurotoxins may be determined by measuring the concentration and activity of AChE, BChE, or both in a subject suspected of being exposed.
As the concentrations and activities of AChE and BChE are affected by certain disease states and exposure to nerve agents, chemical warfare agents, organophosphates (OPs), pesticides, insecticides, anesthetics, and cocaine, it would be desirable to use the concentrations or activities of AChE, BChE, or both, as indicators of a subject""s (1) sensitivity to a drug or chemical, (2) exposure to a nerve agent, a chemical warfare agent, an organophosphate, a pesticide, or insecticide, or (3) disease state.
Unfortunately, the prior art methods for detecting and measuring the concentrations and activities of AChE and BChE are often problematic and inaccurate. Prior art methods have significant drawbacks which include wide statistical error, long clinical turn around times, lack of standardization, the inability to reliably compare results between laboratories, use invasive sampling techniques, are not approved by the United States Food and Drug Administration, use somewhat large blood volumes, and necessitate processing the samples prior to testing, or both. Prior art methods include assays commonly known as gasometric (manometric), Michel, micro-Michel, pH stat, Ellman, and micro-Ellman. These techniques analyze carbon dioxide formation, change in pH, chromophore formation, peroxidase activity, and ultraviolet (UV) absorption. These prior art methods normally determine either the amount of AChE or BChE, but not both simultaneously as red blood cells, plasma, or selective inhibitors are used to measure one or the other. Methods utilizing selective inhibition will not accurately account for samples exposed to certain chemical agents or oximes. Additionally, methods utilizing selective inhibition prevent the simultaneous analysis of AChE and BChE within the same sample, thereby doubling the analysis time and introducing potential errors.
Generally, methods based on gas analysis comprise using acetylcholine as a substrate, bringing acetic acid produced by the enzymatic action of ChE into contact with sodium bicarbonate, and quantitatively determining the carbon dioxide gas produced. This method is problematic as it is cumbersome and difficult to employ high-throughput screening of many samples. Additionally, use of acetylcholine as a substrate is disadvantageous because acetylcholine tends to undergo non-enzymatic hydrolysis and has no high substrate specificity. Furthermore, to achieve greater sensitivity, radioactive sodium bicarbonate has been used which generates regulated waste. This is environmentally unfriendly and increases the cost of the assay.
A pH meter method, like the gas analysis method, comprises using acetylcholine as a substrate, and measuring a pH change due to acetic acid produced by the enzymatic action of ChE by means of a pH meter. The pH meter method suffers from problems similar to the gas method, as well as requiring frequent standardization.
A pH-indicator calorimetric method, unlike the pH meter method, comprises using acetylcholine as a substrate, and measuring a pH change due to acetic acid produced by ChE in terms of the molecular absorbance of the indicator. Indicators utilized include phenol red, bromothymol blue, and m-nitrophenol. Although the pH-indicator calorimetric method may be used to analyze many samples, the reaction time is long, the pH is not kept constant, and the obtained values are not sufficiently reproducible at low and high values.
Assays based on thiocholine color formation utilize acetylthiocholine, butylthiocholine or the like as a substrate. The substrate yields thiocholine by the enzymatic reaction of ChE, which then reacts with 5,5xe2x80x2-dithiobis-2-nitrobenzoic acid (DTNB) to produce a yellow color which is measured by a colorimeter. Although the thiocholine method has a high sensitivity, comprises simple operations, and many samples may be analyzed, it is detrimentally affected by the yellow coloration of bilirubin and hemoglobin in whole blood and is unavoidably affected by compounds having a thiol group such as glutathione. Additionally, the substrate itself is somewhat unstable.
Coupled enzymatic methods utilize benzoylcholine, orthotoluoylcholine or the like as a substrate. These substrate yield betaine by choline oxidase. Then 4-aminoantipyrine is subjected to oxidative condensation with phenol or the like which produces hydrogen peroxide in the presence peroxidase to cause color production. The enzymatic method is problematic since phenol or 4-aminoantipyrine, which is used as the reagent for the color-producing system, competitively inhibits ChE, and the amount of these reagents is limited and sufficient color production is difficult. Additionally, the use of hydrogen peroxide is affected by the presence of bilirubin, reducing substances such as ascorbic acid, and choline. Furthermore, benzoylcholine undergoes non-enzymatic hydrolysis.
One UV method utilizes benzoylcholine as a substrate wherein the decrease in amount of the substrate caused by hydrolysis due to the enzymatic action of ChE at 240 nm is monitored. This UV method is problematic as interference by serum components generally occurs at 240 nm and benzoylcholine undergoes non-enzymatic hydrolysis and the reaction can not be carried out in the optimum pH range of ChE. Additionally, there is a large deviation of absorption coefficient with respect to wavelength.
Another UV method utilizes p-hydroxybenzoylcholine as the substrate wherein p-hydroxybenzoate hydroxylase is reacted with p-hydroxybenzoic acid and the decrease in absorbance caused by the oxidation of NADPH into NADP is monitored at 340 nm. This UV method is problematic as it utilizes NADPH, which is expensive, unstable, must be made frequently, and needs to be kept frozen.
As described above, these conventional methods for determining the ChE activities and concentrations are cumbersome employ reagents and techniques with inherent problems that detrimentally affect precision and accuracy, and are ill suited for high-throughput screening.
There exists a need for an assay and a device for the rapid, accurate and precise detection and measurement of the activity and concentration of at least two proteins, such as AChE and BChE, having similar or overlapping properties towards a plurality of substrates.
In some embodiments, the present invention relates to an assay for detecting, measuring or monitoring the activity or concentration of a protein in a test sample, wherein the protein belongs to a plurality of proteins and the plurality of proteins have similar or overlapping properties towards a plurality of substrates, comprising determining the activity or the concentration of the protein in the test sample with each sensitivity coefficient of each substrate for the protein.
In the embodiments of the invention, the test sample may be a synthetic sample or a natural sample. Natural samples include tissues, fluids, or membranes. Fluids may include blood, serum, lymph, cerebrospinal fluid, breast milk, interstitial or urine. Tissues may include diaphragm, brain, liver, muscle, and kidney.
The sensitivity coefficients are determined from a sensitivity coefficient sample by obtaining a plurality of inhibited dilutions of the sensitivity coefficient sample, wherein the plurality of inhibited dilutions comprise a plurality of concentrations of the protein which are partially to completely inhibited; exposing each inhibited dilution of the plurality of inhibited dilutions to each substrate; measuring the reaction rates between each uninhibited protein in each inhibited dilution and each substrate; calculating the relationships between the reaction rates of each uninhibited protein and each concentration of the sensitivity coefficient sample at infinite inhibitor concentration; and extracting each sensitivity coefficient for each protein from the calculated relationships.
In some embodiments, the plurality of proteins comprise acetylcholinesterase and butyrylcholinesterase. In some embodiments, the plurality of substrates comprise acetylcholine, acetylthiocholine, butyrylcholine, butyrylthiocholine, propionylcholine, and propionylthiocholine. In some embodiments, the inhibitor is huperzine-A, tetraisopropyl pyrophosphoramide, or a combination thereof.
In some embodiments, the invention relates to an assay for detecting, measuring or monitoring the activity or concentration of acetylcholinesterase, butyrylcholinesterase, or both in a test sample comprising determining the activity or the concentration of acetylcholinesterase, butyrylcholinesterase, or both in the test sample with the sensitivity coefficients of each substrate for acetylcholinesterase, butyrylcholinesterase, or both. The plurality of substrates may comprise acetylcholine, acetylthiocholine, butyrylcholine, butyrylthiocholine, propionylcholine, and propionylthiocholine. Preferably, the substrates are acetylthiocholine, butyrylthiocholine, and propionylthiocholine. In these embodiments, the sensitivity coefficients are determined from a sensitivity coefficient sample by obtaining a plurality of dilutions of at least one inhibitor which selectively inhibits either acetylcholinesterase or butyrylcholinesterase; obtaining a plurality of dilutions of the sensitivity coefficient sample; adding each dilution of the inhibitor to each dilution of the sensitivity coefficient sample to obtain a plurality of inhibited sensitivity coefficient samples; exposing each inhibited sensitivity coefficient sample to each substrate; measuring the reaction rates between acetylcholinesterase and each substrate; measuring the reaction rates between butyrylcholinesterase and each substrate; calculating the relationship between the reaction rates of acetylcholinesterase and each concentration of the sensitivity coefficient sample at infinite inhibitor concentration; calculating the relationships between the reaction rates of butyrylcholinesterase and each concentration of the sensitivity coefficient sample at infinite inhibitor concentration; and extracting each sensitivity coefficient of each substrate for acetylcholinesterase and butyrylcholinesterase from the calculated relationships. The inhibitor may be huperzine-A, tetraisopropyl pyrophosphoramide, or a combination thereof. The reaction rates may be measured by utilizing a chromogenic substrate and measuring the absorbance of the reactions.
In some embodiments, the test samples may include an agent which affects the concentration or activity of acetylcholinesterase, butyrylcholinesterase, or both. The agent may be removed from the test sample prior to measuring the reaction rates.
In some embodiments, the present invention relates to a method of detecting or confirming whether a subject was exposed to an agent which affects the concentration or activity of acetylcholinesterase, butyrylcholinesterase, or both comprising obtaining a test sample from the subject; measuring the reaction rates between acetylcholinesterase and a plurality of substrates; measuring the reaction rates between butyrylcholinesterase and the plurality of substrates; and calculating the activity or the concentration of acetylcholinesterase, butyrylcholinesterase, or both with sensitivity coefficients of each substrate for acetylcholinesterase and butyrylcholinesterase.
In some embodiments, the present invention relates to a method of determining the identity of an agent which affects the concentration or activity of acetylcholinesterase, butyrylcholinesterase, or both to which a subject was exposed comprising obtaining a test sample from the subject; measuring the reaction rates between acetylcholinesterase and a plurality of substrates; measuring the reaction rates between butyrylcholinesterase and the plurality of substrates; and calculating the activity or the concentration of acetylcholinesterase, butyrylcholinesterase, or both with sensitivity coefficients of each substrate for acetylcholinesterase and butyrylcholinesterase; and comparing the activities or the concentrations with a database of activity and concentration acetylcholinesterase and butyrylcholinesterase profiles for agents which affect the concentration or activity of acetylcholinesterase, butyrylcholinesterase, or both.
In some embodiments, the present invention relates to a method of determining the efficacy or monitoring the progress of a treatment regime, wherein a subject is administered a compound which affects the concentration or activity of acetylcholinesterase, butyrylcholinesterase, or both comprising obtaining a test sample from the subject; measuring the reaction rates between acetylcholinesterase and a plurality of substrates; measuring the reaction rates between butyrylcholinesterase and the plurality of substrates; and calculating the activity or the concentration of acetylcholinesterase, butyrylcholinesterase, or both with sensitivity coefficients of each substrate for acetylcholinesterase and butyrylcholinesterase; and monitoring the activities or the concentrations of acetylcholinesterase, butyrylcholinesterase, or both as a function of time of the treatment regime.
In some embodiments, the present invention relates to a method of determining whether a subject suffers from a drug sensitivity or a disease which affects the activities or the concentrations of acetylcholinesterase, butyrylcholinesterase, or both comprising obtaining a test sample from the subject; measuring the reaction rates between acetylcholinesterase and a plurality of substrates; measuring the reaction rates between butyrylcholinesterase and the plurality of substrates; and calculating the activity or the concentration of acetylcholinesterase, butyrylcholinesterase, or both with sensitivity coefficients of each substrate for acetylcholinesterase and butyrylcholinesterase; and comparing the activities or the concentrations with a database of activity and concentration acetylcholinesterase and butyrylcholinesterase profiles which are typical of individuals suffering from given drug sensitivities and individuals suffering from given diseases which affect the activities or the concentrations of acetylcholinesterase, butyrylcholinesterase, or both.
In some embodiments, the present invention relates to a method of measuring the concentration of red blood cells in a subject comprising obtaining a test sample from the subject; measuring the reaction rates between acetylcholinesterase and a plurality of substrates; measuring the reaction rates between butyrylcholinesterase and the plurality of substrates; and calculating the activity or the concentration of acetylcholinesterase, butyrylcholinesterase, or both with sensitivity coefficients of each substrate for acetylcholinesterase and butyrylcholinesterase; determining a relationship between standard concentrations of red blood cells and the activities or the concentrations of acetylcholinesterase, butyrylcholinesterase, or both; and using the relationship to calculate the concentration of red blood cells of the sample.
In some embodiments, the present invention relates to a method of screening for a candidate compound which affects the concentration or activity of acetylcholinesterase, butyrylcholinesterase, or both comprising obtaining a test sample; measuring the reaction rates between acetylcholinesterase and a plurality of substrates; measuring the reaction rates between butyrylcholinesterase and the plurality of substrates; and calculating the activity or the concentration of acetylcholinesterase, butyrylcholinesterase, or both with sensitivity coefficients of each substrate for acetylcholinesterase and butyrylcholinesterase; and determining whether the concentration or activity of acetylcholinesterase, butyrylcholinesterase, or both changes.
In some embodiments, the present invention relates to a device for detecting, measuring or monitoring the activities or concentrations of acetylcholinesterase, butyrylcholinesterase, or both in a test sample wherein the device measures the reaction rates between acetylcholinesterase and butyrylcholinesterase and at least two substrates; and calculates the activities or the concentrations of acetylcholinesterase, butyrylcholinesterase, or both with sensitivity coefficients of each substrate for acetylcholinesterase and butyrylcholinesterase. The device may further comprise a cartridge comprising the reagents, buffers, substrates and standards for measuring the reaction rates.
In some embodiments, the present invention relates to a kit for detecting, measuring or monitoring the activities or concentrations of acetylcholinesterase, butyrylcholinesterase, or both in a test sample comprising substrates for acetylcholinesterase and butyrylcholinesterase. The kit may further comprise a device for measuring the reaction rates between acetylcholinesterase and butyrylcholinesterase and the substrates, and calculating the activities or concentrations acetylcholinesterase and butyrylcholinesterase. The substrates for acetylcholinesterase and butyrylcholinesterase may include acetylthiocholine, butyrylthiocholine, and propionylthiocholine. The kit may also include a chromogenic substrate. The kit may also include directions.
In some embodiments, the present invention relates to a biosensor capable of detecting an agent which affects the concentration or activity of acetylcholinesterase, butyrylcholinesterase, or both wherein the comprises a known mixture of acetylcholinesterase and butyrylcholinesterase immobilized on a support and a sealed chamber containing the known mixture of acetylcholinesterase and butyrylcholinesterase.
In some embodiments, the present invention relates to a database of sensitivity coefficients for calculating the activities or the concentrations of acetylcholinesterase, butyrylcholinesterase, or both made by a method comprising obtaining a plurality of inhibited dilutions of a sensitivity coefficient sample, wherein the plurality of inhibited dilutions comprise a plurality of concentrations of either acetylcholinesterase or butyrylcholinesterase which is partially to completely inhibited; exposing each inhibited dilution of the plurality of inhibited dilutions to each substrate in a plurality of substrates for acetylcholinesterase and butyrylcholinesterase; measuring the reaction rates between acetylcholinesterase and each substrate; measuring the reaction rates between butyrylcholinesterase and each substrate; calculating the relationship between the reaction rates of acetylcholinesterase and each concentration of the sensitivity coefficient sample at infinite inhibitor concentration; calculating the relationships between the reaction rates of butyrylcholinesterase and each concentration of the sensitivity coefficient sample at infinite inhibitor concentration; and extracting each sensitivity coefficient of each substrate for acetylcholinesterase and butyrylcholinesterase from the calculated relationships.